2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 10th 2017 to January 22nd 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD guideline 209 was followed

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

OECD 209 for Testing of Chemicals (2010)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate of Noack laboratorien GmbH : inspection between June 7th and 10th and on July 13th 2016.

Specific details on test material used for the study:

- Storage condition of test material: Storage in closed, preferably full containers, away from heat sources and protected from extreme variations in temperature.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The reference item concentration was diluted out of a stock solution.
- Controls: Triplicates of the control without test item were included at the beginning and at the end of the test.

Test organisms (species):

activated sludge

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant of 31137 Hildesheim, Germany
- Preparation of inoculum for exposure: A fresh sample of activated sludge taken from the sewage plant Hildesheim was used for the test procedure. It was washed twice and adjusted with chlorine free tap water if necessary to a dry sludge concentration of 3.0 g/L ± 10 %.
- The sludge was used within 24 h after sampling.
- Pretreatment: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Dry sludge concentration : 3.16 g/L, corresponding to 1.58 g/L in the test vessel

Test type:

static

Water media type:

other: Demineralised water

Limit test:

no

Total exposure duration:

3 h

Test temperature:

19.9 °C

pH:

pH-value of the activated sludge : 7.83, pH-value of the synthetic waste water: 7.28

Dissolved oxygen:

Kept above 60-70 %

Nominal and measured concentrations:

Nominal concentrations : 10 - 32 - 100 - 320 - 1000 mg/L were weighed out on object slides and were transferred to laboratory bottles.

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 mL laboratory bottles, ISO 4796

- Aeration: Shaking of the laboratory bottles at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.

- No. of vessels per concentration (replicates): 5 replicates

- No. of vessels per control (replicates): 3 replicates

- Sludge concentration (weight of dry solids per volume): 3.16 g/L

- Nutrients provided for bacteria: Synthetic waste water according to OECD Guideline 209

- Nitrification inhibitor used (delete if not applicable): none

OTHER TEST CONDITIONS
- Adjustment of pH: No

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : inhibition of respiration

TEST CONCENTRATIONS
- Spacing factor for test concentrations: The definitive study was carried out with 5 concentrations within the range 10 - 1000 mg/L test item in a geometrical series and with a dilution factor of 3.2.
- Range finding study : A preliminary test (non GLP) was carried out with the concentrations of the test substance of 10 – 100 - 1000 mg/L with two replicates each.
- Test item concentrations (definitive test) : 10 - 32 - 100 - 320 - 1000 mg/L.
- Results used to determine the conditions for the definitive study: the inhibitions registered in the preliminary test were:
5 and 7 % for 10 mg test item/L
6 and 7 % for 100 mg test item/L
9 and 13 % for 1000 mg test item/L

Reference substance (positive control):

yes

Remarks:

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined.

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

The EC50-value for the reference item was 102 mg/L.

Reported statistics and error estimates:

When running a One Way Analysis of Variance a normality test and an Equal Variance test were done first. P-value for both tests was 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha=0.05.

Table 6.1.7/1: Oxygen uptake rates and specific respiration rates of the control replicates

	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Specific Respiration Rate Rs
		[mg O2/L]	[mg O2/(L×h)]	[mg O2/g×h]
Control	1	7.67	31.9	20.2
	2	7.81	32.7	20.7
	3	7.43	29.9	18.9
	4	7.29	31.4	19.9
	5	7.30	31.0	19.6
	6	7.21	28.9	18.3
	Mean value		31.0	19.6
	Standard deviation		1.26
	CV [%]		4.1

(*) determined at start of the measurement

Repl. = Replicate

CV = Coefficient of variation

Table 6.1.7/2: Oxygen uptake rates and inhibition of the respiration of the test item concentrations

Test Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
10	1	5.81	30.8	1	8
	2	6.01	30.2	3
	3	5.69	28.6	8
	4	6.84	28.7	7
	5	5.16	24.5	21
32	1	6.08	30.3	2	6
	2	6.10	31.0	0
	3	5.79	28.8	7
	4	6.97	29.1	6
	5	5.22	26.5	15
100	1	5.81	30.8	1	8
	2	5.96	30.9	0
	3	6.17	28.9	7
	4	11.9	25.4	18
	5	5.72	25.9	16
320	1	6.39	30.2	3	9
	2	6.42	29.8	4
	3	6.07	27.4	12
	4	6.55	29.8	4
	5	5.32	24.9	20
1000	1	5.54	28.9	7	10
	2	5.58	28.9	7
	3	5.04	27.8	10
	4	6.03	29.4	5
	5	6.97	25.1	19

(*) determined at start of the measurement

Repl. = Replicate

Table 6.1.7/3: EC-Values with Confidence Interval of the test substance

	EC-values [mg/L]	Confidence interval P = 95 [%] [mg/L]
NOEC*	1000	̶
EC10	> 1000	–
EC20	> 1000	–
EC50	> 1000	–
EC80	> 1000	-

*) No statistically significant inhibition (P=0.211), ANOVA, Dunnett`s Method

Table 6.1.7/4: Oxygen Uptake Rates and Inhibition of the Respiration of the Reference Item Concentrations

Reference Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
58	1	7.68	23.8	23	27
	2	8.07	22.9	26
	3	7.55	21.5	31
100	1	8.17	17.5	44	46
	2	8.19	17.3	44
	3	7.66	15.6	50
180	1	8.77	7.6	75	77
	2	8.59	7.4	76
	3	8.24	6.5	79

(*) determined at start of the measurement

Repl. = Replicate

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test substance is 1000 mg/L. The EC10 is > 1000 mg/L and the EC50 is > 1000 mg/L.

Executive summary:

A respiration inhibition test with activated sludge according to OECD Guideline No. 209 was carried out with the test substance.

The test was carried out under static conditions with the test item concentrations 10 - 32 - 100 - 320 - 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 102 mg/L. The NOEC of the test substance is 1000 mg/L. The EC10 is > 1000 mg/L and the EC50 is > 1000 mg/L.

Description of key information

OECD Guideline 209, GLP, key study, validity 1:

3h-NOEC = 1000 mg/L

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

A respiration inhibition test with activated sludge according to OECD Guideline No. 209 was carried out with the registered substance.

The test was carried out under static conditions with the test item concentrations 10 - 32 - 100 - 320 - 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 102 mg/L.The NOEC of the test substance is 1000 mg/L. The EC10 is > 1000 mg/L and the EC50 is > 1000 mg/L.